Rotating Anode X-ray Source Research Articles

Breakthroughs in understanding elastic grain interaction and whisker formation made possible by advances in X-ray powder diffraction

Recent work on laboratory parallel-beam diffraction is summarized. Special attention is paid to instrumental aberrations and their corrections. Two types of primary beam optics are considered: X-ray lenses and X-ray multilayer mirrors. A high photon flux, high brilliance and minimal divergence of the incident beam make a recently pioneered setup, based on a combination of a microfocus rotating anode X-ray source with a single-reflection mirror, particularly suitable for grazing-incidence diffraction, and thus for the investigation of very thin films (yielding low diffracted intensities) and of stress and the anisotropy of the microstructure of materials (such as the crystallographic texture, requiring the acquisition of large measured data sets, corresponding to the variation of the orientation of the diffraction vector with respect to the specimen frame of reference). A comparative discussion of primary optics which can be used to realize parallel-beam geometry demonstrates the range of possible applications of parallel-beam diffractometers. Such advances in laboratory parallel-beam diffraction instrumentation enable highly accurate diffraction investigations of thin films and, in particular, diffraction measurement at constant, tuneable penetration depth. These novel possibilities thereby provide a pathway for fundamental investigations of thin films and nanocrystalline materials. In particular, breakthroughs have been realized in the fields of elastic grain interaction in ultrathin films and the understanding of whisker formation on Sn coatings.

The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.

ChemInform Abstract: Polycitone A and Polycitrins A and B: New Alkaloids from the Marine Ascidian Polycitor sp..

Three novel compound polycitone A (1a) and polycitrins A and B (2 and 3) were isolated from the marine ascidian Polycitor sp. The structures of compounds 1a, 2, and 3 were established mainly on the basis of NMR spectroscopic data and, in the case of 1a, also by single-crystal X-ray diffraction analysis. The crystallographic analysis was performed on 1b, the penta-O-methyl derivative of 1a, both compound yielding poorly diffracting crystals of highly anisotropic shape. It required, in view of the dominating heavy-atom content of 1b, diffraction measurements with an intense rotating-anode X-ray source. Polycitone A and polycitrins A and B represent the first examples of two new classes of marine products which might biogenetically be close to the lamellarins. The penta-O-methyl derivatives 1b, was found to inhibit the growth of SV40 transformed fibroblast cells in a concentration of 10 μg/mL

Crystallization and preliminary crystallographic analysis of recombinant VSP1 from Arabidopsis thaliana.

VSP1 is a defence protein in Arabidopsis thaliana that may also be involved in control of plant development. The recombinant protein has been overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 1.9 A resolution and a complete X-ray data set was collected at 100 K using Cu Kalpha radiation from a rotating-anode X-ray source. The crystals belonged to space group C2. As there are no related structures that could be used as a search model for molecular replacement, work is in progress on experimental phasing using heavy-atom derivatives and selenomethionine derivatives.

Crystallization and Preliminary X-Ray Diffraction Analysis of ARO9, an Aromatic Aminotransferase from Saccharomyces cerevisiae

Saccharomyces cerevisae ARO9 protein, an aromatic aminotransferase II, catalyzes the transamination step of the catabolism of aromatic amino acids, mainly tryptophan. ARO9 also belongs to a novel subfamily of enzymes within the aminotransferase subgroup I. Crystals of ARO9 protein have been grown using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 75.6 A, b = 117.5 A, c = 134.9 A. Diffraction data were collected to a resolution of 2.6 A using a rotating-anode X-ray source. Analysis indicates the presence of two molecules in an asymmetric unit.

High-resolution X-ray imaging—a powerful nondestructive technique for applications in semiconductor industry

The availability of high-brilliance X-ray sources, high-precision X-ray focusing optics and very efficient CCD area detectors has contributed essentially to the development of transmission X-ray microscopy (TXM) and X-ray computed tomography (XCT) with sub-50 nm resolution. Particularly, the fabrication of high aspect ratio Fresnel zone plates with zone widths approaching 15 nm has contributed to the enormous improvement in spatial resolution during the previous years. Currently, Fresnel zone plates give the ability to reach spatial resolutions of 15 to 20 nm in the soft and of about 30 to 50 nm in the hard X-ray energy range. X-ray microscopes with rotating anode X-ray sources that can be installed in an analytical lab next to a semiconductor fab have been developed recently. These unique TXM/XCT systems provide an important new capability of nondestructive 3D imaging of internal circuit structures without destructive sample preparation such as cross sectioning. These lab systems can be used for failure localization in micro- and nanoelectronic structures and devices, e.g., to visualize voids and residuals in on-chip metal interconnects without physical modification of the chip. Synchrotron radiation experiments have been used to study new processes and materials that have to be introduced into the semiconductor industry. The potential of TXM using synchrotron radiation in the soft X-ray energy range is shown for the nondestructive in situ imaging of void evolution in embedded on-chip copper interconnect structures during electromigration and for the imaging of different types of insulating thin films between the on-chip interconnects (spectromicroscopy).

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa.

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 A, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 A resolution on a rotating-anode X-ray source.

Purification, crystallization and preliminary crystallographic analysis of the non-Pfam protein AF1514 from Archeoglobus fulgidus DSM 4304.

A 10.5 kDa non-Pfam hypothetical protein, AF1514, from the hyperthermophilic archaeon Archeoglobus fulgidus has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 2.09 A resolution and a data set was collected at 100 K using Cu K alpha radiation from a rotating-anode X-ray source. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 49.27, c = 106.61 A. The calculated Matthews coefficient was 3.16 A(3) Da(-1), suggesting the presence of one molecule in the asymmetric unit.

Application of a single-reflection collimating multilayer optic for X-ray diffraction experiments employing parallel-beam geometry

Instrumental aberrations of a parallel-beam diffractometer equipped with a rotating anode X-ray source, a single-reflection collimating multilayer optic and a parallel-plate collimator in front of the detector have been investigated on the basis of standard measurements (i.e.employing stress- and texture-free isotropic powder specimens exhibiting small or negligible structural diffraction line broadening). It has been shown that a defocusing correction, which is a major instrumental aberration for diffraction patterns collected with divergent-beam (focusing) geometries, is unnecessary for this diffractometer. The performance of the diffractometer equipped with the single-reflection collimating multilayer optic (single-reflection mirror) is compared with the performance of the diffractometer equipped with a Kirkpatrick–Baez optic (cross-coupled Göbel mirror) on the basis of experimental standard measurements and ray-tracing calculations. The results indicate that the use of the single-reflection mirror provides a significant gain in photon flux and brilliance. A high photon flux, high brilliance and minimal divergence of the incident beam make the setup based on the single-reflection mirror particularly suitable for grazing-incidence diffraction, and thus for the investigation of very thin films (yielding low diffracted intensities) and of stress and texture (requiring the acquisition of large measured data sets, corresponding to the variation of the orientation of the diffraction vector with respect to the specimen frame of reference). A comparative discussion of primary optics which can be used to realise parallel-beam geometry shows the range of possible applications of parallel-beam diffractometers and indicates the virtues and disadvantages of the different optics.

Analyzer-based phase contrast imaging and phase retrieval using a rotating anode x-ray source

We have performed an analyzer crystal based phase contrast imaging (ABI) experiment using a rotating anode x-ray source. The use of such an incoherent source demonstrates the potential of ABI as a quantitative characterization tool for the laboratory environment. A phase contrast image of a plastic phantom was recorded on high resolution x-ray film and the projected thickness was retrieved from a single image. The projected thickness recovered from the phase contrast image was shown to quantitatively agree with a reference optical microscope measurement.

X-ray computed tomography in Zernike phase contrast mode at 8 keV with 50-nm resolution using Cu rotating anode X-ray source

High-resolution X-ray computed tomography (XCT) enables nondestructive 3D imaging of complex structures, regardless of their state of crystallinity. This work describes a sub-50 nm resolution XCT system operating at 8 keV in absorption and Zernike phase contrast modes based on a commercially available Cu rotating anode laboratory X-ray source. The system utilizes a high efficiency reflective capillary condenser lens and high-resolution Fresnel zone plates with an outermost zone width of 35 nm and 700 nm structure height resulting in a spatial resolution better than 50 nm currently. Imaging a fragment of the solid oxide fuel cells (SOFC) with 50 nm resolution is presented as an application example of the XCT technique in materials science and nanotechnology.

Crystallization and preliminary X-ray crystallographic analysis of BxlE, a xylobiose transporter from Streptomyces thermoviolaceus OPC-520.

Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 44.63, b = 63.27, c = 66.40 A, beta = 103.05 degrees, and contained one 48 kDa molecule per asymmetric unit (V(M) = 1.96 A3 Da(-1)). Diffraction data collected to a resolution of 1.65 A using a rotating-anode X-ray source gave a data set with an overall R(merge) of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method.

Focus on nanoscience and nanotechnology

Focus on nanoscience and nanotechnology

Characterization of a polycapillary focusing X-ray lens for application in spatially resolved EXAFS experiments

A dispersive extended-X-ray-absorption-fine-structure (EXAFS) spectrometer based on a polycapillary focusing X-ray lens (PFXRL), a position-sensitive proportional counter and a rotating anode X-ray source is designed. When the working voltage and current of a Mo rotating anode X-ray generator are 25 kV and 100 mA, respectively, it takes 6 h to obtain the EXAFS spectrum of the Cu film. The experiments show that a dispersive EXAFS spectrometer based on a PFXRL can be applied in spatially resolved EXAFS analysis.

Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase fromLeptospira interrogans

2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 A resolution using a Cu K alpha rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 A, beta = 100.9 degrees. The V(M) is calculated to be 2.4 A3 Da(-1), assuming there to be 12 protein molecules in the asymmetric unit.

Expression, purification and preliminary crystallographic analysis of theToxoplasma gondiienoyl reductase

The protozoan parasite Toxoplasma gondii is the causative agent of one of the most widespread parasitic infections of man and is a leading cause of congenital neurological birth defects and the third most common cause of food-borne deaths in the United States. Despite this, to date no drugs are available that provide a fully effective treatment. Recently, the antibacterial agent triclosan was shown to inhibit the fatty-acid biosynthesis pathway in T. gondii and to interact with the enoyl reductase (ENR). In order to analyse the potential of triclosan as a lead compound targeting T. gondii ENR and to explore unique features of the apicomplexan enzyme that could be exploited in future drug development, structural studies have been initiated on T. gondii ENR. Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant. The crystals belong to space group P3(2)21, with approximate unit-cell parameters a = 78.1, b = 78.1, c = 188.5 A, alpha = beta = 90, gamma = 120 degrees and a dimer in the asymmetric unit. Test data were collected to beyond 2.6 A on cryocooled crystals (100 K) using a Rigaku MM007 rotating-anode X-ray source, revealing that the crystals are suitable for a full structural determination.

Dehydrogenation kinetics of pure and nickel-doped magnesium hydride investigated by in situ time-resolved powder X-ray diffraction

The dehydrogenation kinetics of pure and nickel (Ni)-doped (2 w/w%) magnesium hydride ( MgH 2 ) have been investigated by in situ time-resolved powder X-ray diffraction (PXD). Deactivated samples, i.e. air exposed, are investigated in order to focus on the effect of magnesium oxide (MgO) surface layers, which might be unavoidable for magnesium (Mg)-based storage media for mobile applications. A curved position-sensitive detector covering 120 ∘ in 2 θ and a rotating anode X-ray source provide a time resolution of 45 s and up to 90 powder patterns collected during an experiment under isothermal conditions. A quartz capillary cell allowed the in situ study of gas/solid reactions. Three phases were identified: Mg, MgH 2 and MgO and their phase fractions were extracted by Rietveld refinement or integration of selected reflections from each phase. Dehydrogenation curves were constructed and analysed by the Johnson–Mehl–Avrami formalism in order to derive rate constants at different temperatures. The apparent activation energies for dehydrogenation of pure and Ni-doped magnesium hydride were E A ≈ 300 and 250 kJ / mol , respectively. Differential scanning calorimetry gave, E A = 270 kJ / mol for dehydrogenation of the Ni-doped sample. The relatively high activation energies are due to MgO surface layers, retarding the diffusion of hydrogen ( H 2 ) out of MgH 2 / Mg . The observed difference in E A of ca. 50 kJ/mol is likely due to the catalytic effect of Ni on the recombination of H atoms to H 2 molecules verified by theoretical considerations.

Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein fromMoorella thermoacetica

A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.

Crystallization and preliminary X-ray studies of mouse centrin1

Centrins belong to a family of Ca2+-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure-function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW approximately 22 240 Da) could be crystallized at room temperature, using 20-25%(w/v) PEG 1500, 5-10%(v/v) ethylene glycol and 1-2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, beta = 109.4 degrees. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 A3 Da(-1).

Structural Basis for Metal Ion Coordination and the Catalytic Mechanism of Sphingomyelinases D

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-angstrom resolution using the "quick cryo-soaking" technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (alpha/beta)8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu32, Asp34, Asp91, and solvent molecules. In the proposed acid base catalytic mechanism, His12 and His47 play key roles and are supported by a network of hydrogen bonds between Asp34, Asp52, Trp230, Asp233, and Asn252.